The movement of cells during migration, motility, chemotaxis or wound healing, or the movement of small organisms such as zebrafish or nematodes is an important parameter in the study of biological systems. For example, many studies are directed at measuring the response of cells or cell cultures to a physical stimulus or a chemical treatment. Toxicological studies, in particular, often focus on the response of cell cultures to chemical treatment to determine if a chemical has an adverse effect on the growth and development of the test or cultured organism.
Cardiotoxicity currently accounts for 30% of drug failures during pre-clinical and clinical development and there is a strong demand from the pharmaceutical industry for more predictive cellular models to reduce attrition costs. Cardiomyocytes derived from human embryonic stem cells provide an advance towards development of more clinically predictive assays for assessing cardiotoxicity liabilities in new drug candidates. Cardiomyocytes may be used in a wide range of applications including electrophysiology, ion flux imaging and high content analysis to assess cardiac liability of candidate drugs. Cardiomyocyte function is controlled by an integrated system of ion channels which modulate the influx and efflux of potassium, calcium and sodium ions to modulate cellular contractility. Drug interference with these control mechanisms, e.g. via interaction with the HERG potassium channel, can lead to shortening or lengthening of cardiomyocyte action potentials and in some cases to early or late after-depolarisations which in-vivo may give rise to arrhythmia and heart failure.
Measurement of cardiomyocyte beat rate is a commonly used technique to assess drug cardiac liability. Cultured cardiomyocytes are imaged by video microscopy and video edge detection techniques are used to measure the rate at which the edge of a cell or cluster of cells moves into and out of a user determined detection zone (Gervais-Pingot et. al. 1994 Cell Biol Toxicol. 10(5-6):297-300; Dolnikov et. al. 2006 Stem Cells. 4(2):236-45.). This method requires dedicated equipment including specialised electronic hardware to perform video rate edge detection, for example VED motion edge detectors (www.crescent-electronics.com). Since the method relies on detection of movement of an object edge with high contrast the method is not suitable for all cultures, particularly those with high cell density where the imaged area is full of cells. Moreover since the technique requires an operator to establish the region of analysis for edge detection for each sample to be analysed the approach cannot be implemented in high throughput.
US 2008/0304732 describes methods for evaluation of cellular motion applied to cardiomyocyte cultures wherein time series images are acquired and motion vectors are derived for successive sequential pairs of images through the time series. These motion vectors are based on the displacement of each individual cell between consecutive image pairs and cellular motion is represented by a series of displacement vector diagrams indicating the presence and direction of movement between successive images. Cellular motion data are then extracted using optical flow algorithms and the resulting complex data reduced or decomposed using factorisation methods, such as principal component analysis, to allow motion data to be represented in low dimensional space, for example as a waveform plot. These methods require significant computer processing power and time to perform complex image and data analysis.
The present invention seeks to overcome the limitations of prior art methods by providing a system and a method of determining motion of a biological object using simple image subtraction techniques which are independent of cell density, image contrast or the presence of detectable edges. Furthermore, the present invention does not require complex data reduction or decomposition techniques, and may be implemented using automated high-throughput imaging equipment.